Ex Vivo Colonic Tissue Susceptibility to HIV-1 in Cisgender Men and Women.

The biology of HIV-1 acquisition through unprotected receptive anal intercourse is understudied. Considering that sex hormones are implicated in intestinal physiology, pathology, and HIV acquisition and pathogenesis, we explored links between sex hormones, ex vivo HIV-1BaL infection of colonic mucosa, and candidate biomarkers of susceptibility to HIV-1 (CD4+ T cell frequencies and immune mediators) in cisgender women and men. No consistent significant associations between sex hormone concentrations and ex vivo tissue infection with HIV-1BaL were detected. In men, serum estradiol (E2) concentrations were positively associated with tissue proinflammatory mediators (IL17A, GM-CSF, IFNγ, TNFα, and MIG/CXCL9) and serum testosterone concentrations were negatively associated with frequencies of activated CD4+ T cells (CD4+CCR5+, CD4+HLA-DR+, and CD4+CD38+HLA-DR+). In women, the only significant interactions were positive associations between progesterone (P4)/E2 ratios and tissue ILRA concentrations and between P4/E2 ratios and frequencies of tissue CD4+α4ß7high+ T cells. The study did not reveal relationships between biological sex or phase of the menstrual cycle and ex vivo tissue HIV-1BaL infection and tissue immune mediators. A comparison of CD4+ T cell frequencies between study groups revealed a higher frequency of tissue CD4+α4ß7high+ T cells in women versus men. In contrast, higher frequencies of tissue CD4+CD103+ T cells were detected in men versus women in the follicular phase of the menstrual cycle. Overall, the study identified associations between systemic sex hormone concentrations, biological sex, and tissue candidate biomarkers of susceptibility to HIV-1. The significance of these results for tissue susceptibility to HIV-1 and early HIV-1 pathogenesis warrants further investigation.

New approaches for developing biomarkers of hormonal contraceptive use.

To identify biomarkers of hormonal contraceptive (HC) use in urine and saliva, we conducted a pilot study with 30 women initiating levonorgestrel (LNG) containing combined oral contraceptives (COCs) or depot medroxyprogesterone acetate (DMPA) (15/group). Based on established COC pharmacokinetics, we collected serum and urine samples before COC ingestion and during Days one and three of use, or before DMPA injection and on Days 21 and 60 post-injection. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure serum/urine LNG and MPA. LNG was undetectable at baseline (specificity 100%); post ingestion, most urine samples had detectable LNG levels (sensitivity: 80% 6 h post Dose one, 93% 6 h post Dose three). We used a DetectX LNG immunoassay kit and showed 100% sensitivity measuring urine LNG. Urine MPA levels were undetectable in 14/15 women at baseline (specificity 91%); post-injection all urine samples had detectable MPA levels (sensitivity: 100% days 21 and 60). Results suggest urine sampling can be used to identify a biomarker of LNG and MPA use. Based on evidence from other steroidal hormonal studies showing changes affecting the transcriptome profile of saliva at 24 h, we used the same (COC, DMPA) timepoints to collect saliva. We performed transcriptome analysis and detected several differentially expressed genes in DMPA users' saliva on Days 21 and 60 compared to baseline; none among COC users. We plan further research of differential gene expression in saliva as a HC biomarker of DMPA use, and will explore longer periods of COC use and saliva collection times, and application of microRNA sequencing to support using saliva as a COC biomarker.

Results of a phase 1, randomized, placebo-controlled first-in-human trial of griffithsin formulated in a carrageenan vaginal gel.

HIV pre-exposure prophylaxis (PrEP) is dominated by clinical therapeutic antiretroviral (ARV) drugs. Griffithsin (GRFT) is a non-ARV lectin with potent anti-HIV activity. GRFT's preclinical safety, lack of systemic absorption after vaginal administration in animal studies, and lack of cross-resistance with existing ARV drugs prompted its development for topical HIV PrEP. We investigated safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PC-6500 (0.1% GRFT in a carrageenan (CG) gel) in healthy women after vaginal administration. This randomized, placebo-controlled, parallel group, double-blind first-in-human phase 1 study enrolled healthy, HIV-negative, non-pregnant women aged 24-45 years. In the open label period, all participants (n = 7) received single dose of PC-6500. In the randomized period, participants (n = 13) were instructed to self-administer 14 doses of PC-6500 or its matching CG placebo (PC-535) once daily for 14 days. The primary outcomes were safety and PK after single dose, and then after 14 days of dosing. Exploratory outcomes were GRFT concentrations in cervicovaginal fluids, PD, inflammatory mediators and gene expression in ectocervical biopsies. This trial is registered with ClinicalTrials.gov, number NCT02875119. No significant adverse events were recorded in clinical or laboratory results or histopathological evaluations in cervicovaginal mucosa, and no anti-drug (GRFT) antibodies were detected in serum. No cervicovaginal proinflammatory responses and no changes in the ectocervical transcriptome were evident. Decreased levels of proinflammatory chemokines (CXCL8, CCL5 and CCL20) were observed. GRFT was not detected in plasma. GRFT and GRFT/CG in cervicovaginal lavage samples inhibited HIV and HPV, respectively, in vitro in a dose-dependent fashion. These data suggest GRFT formulated in a CG gel is a safe and promising on-demand multipurpose prevention technology product that warrants further investigation.

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo.

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.

Development of a Vaginal Fast-Dissolving Insert Combining Griffithsin and Carrageenan for Potential Use Against Sexually Transmitted Infections.

Precoital, on-demand topical microbicides to reduce a woman's risk of sexually transmitted infections have been in development for nearly 3 decades, but no product has been approved due to acceptability issues and poor adherence in clinical trials. We set out to develop a self-administered vaginal fast-dissolving insert (FDI) produced by freeze-drying that would deliver safe and effective amounts of the antiviral agents griffithsin (GRFT) and carrageenan (CG) and would have properties women and their partners find acceptable. We evaluated FDI physical criteria, attributes of the gel produced upon dissolving, and GRFT stability. The lead formulation, FDI-024, was selected from 13 candidates and contains 4 mg of GRFT, 15 mg of CG, and excipients (the cryoprotectant sucrose and bulking agents dextran 40 and mannitol). The FDI exhibits good friability and hardness and is stable for at least 6 months at up to 40°C/75% relative humidity. It disintegrates in less than 60 s in a physiologically relevant volume (∼1 mL) of simulated vaginal fluid, forming a viscous semi-solid gel with favorable mucoadhesive and spreading properties. The formulation retains the antiviral activity of GRFT and CG against HIV type 1 and human papillomavirus, respectively, in cell-based assays.

An intravaginal ring that releases three antiviral agents and a contraceptive blocks SHIV-RT infection, reduces HSV-2 shedding, and suppresses hormonal cycling in rhesus macaques.

Women globally need access to multipurpose prevention technologies (MPTs) that prevent human immunodeficiency virus (HIV), sexually transmitted infections that increase HIV acquisition/transmission risk, and unintended pregnancy. Seeking an MPT with activity against HIV, herpes simplex virus-2 (HSV-2), and human papillomavirus (HPV), we developed a prototype intravaginal ring (IVR), the MZCL IVR, which released the antiviral agents MIV-150, zinc acetate, and carrageenan (MZC for short) and the contraceptive levonorgestrel (LNG). Previously, we showed that an MZC gel has potent activity against immunodeficiency viruses, HSV-2, and HPV and that the MZCL (MZC with LNG) IVR releases all four components in macaques in vivo at levels associated with efficacy. Vaginal fluid from treated macaques has in vitro activity against HIV, HSV-2, and HPV. Herein, we assessed the ability of the MZCL IVR to protect macaques against repeated co-challenge with HSV-2 and SHIV-RT (simian immunodeficiency virus [SIV] containing the reverse transcriptase gene from HIV) and prevent hormonal cycling. We evaluated in vivo drug release in co-challenged macaques by measuring drug levels in blood and vaginal fluid and residual drug levels in used IVRs. The MZCL IVR significantly prevented SHIV-RT infection, reduced HSV-2 vaginal shedding, and prevented cycling. No non-nucleoside HIV reverse transcriptase inhibitor (NNRTI)-resistant SHIV was detected in macaques that became infected after continuous exposure to MZC from the IVR. Macaques wearing the MZCL IVR also had carrageenan levels in vaginal fluid expected to protect from HPV (extrapolated from mice) and LNG levels in blood associated with contraceptive efficacy. The MZCL IVR is a promising MPT candidate that warrants further development.

A Novel Microbicide/Contraceptive Intravaginal Ring Protects Macaque Genital Mucosa against SHIV-RT Infection Ex Vivo.

Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.

Multipurpose Prevention Approaches with Antiretroviral-Based Formulations.

We compared the preclinical safety and efficacy of tenofovir (TFV) 1% gel with that of MZC gel [containing 50 µM MIV-150, 14 mM Zn(O2CCH3)2(H2O)2, and 3% carrageenan] through a series of in vitro, ex vivo, and in vivo assays. The two gels showed good antiviral therapeutic indexes (50% cytotoxic concentration/50% effective concentration ratios; range, >25 to 800). MZC showed greater anti-simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) activity than TFV 1% gel in rhesus macaque vaginal explants. MZC protected mice from vaginal herpes simplex virus 2 (HSV-2) challenge (P < 0.0001), but the TFV 1% gel did not.

Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus.

Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC50 = 0.429 to 1.39 µM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P < 0.026), greater CG-mediated protection was afforded by the GRFT-CG combination for up to 8 h (P < 0.0022). These findings support the development of the GRFT-CG combination as a broad-spectrum microbicide.

A novel intravaginal ring to prevent HIV-1, HSV-2, HPV, and unintended pregnancy.

Women urgently need a self-initiated, multipurpose prevention technology (MPT) that simultaneously reduces their risk of acquiring HIV-1, HSV-2, and HPV (latter two associated with increased risk of HIV-1 acquisition) and prevents unintended pregnancy. Here, we describe a novel core-matrix intravaginal ring (IVR), the MZCL IVR, which effectively delivered the MZC combination microbicide and a contraceptive. The MZCL IVR contains four active pharmaceutical ingredients (APIs): MIV-150 (targets HIV-1), zinc acetate (ZA; targets HIV-1 and HSV-2), carrageenan (CG; targets HPV and HSV-2), and levonorgestrel (LNG; targets unintended pregnancy). The elastomeric IVR body (matrix) was produced by hot melt extrusion of the non-water swellable elastomer, ethylene vinyl acetate (EVA-28), containing the hydrophobic small molecules, MIV-150 and LNG. The solid hydrophilic core, embedded within the IVR by compression, contained the small molecule ZA and the macromolecule CG. Hydrated ZA/CG from the core was released by diffusion via a pore on the IVR while the MIV-150/LNG diffused from the matrix continuously for 94 days (d) in vitro and up to 28 d (study period) in macaques. The APIs released in vitro and in vivo were active against HIV-1ADA-M, HSV-2, and HPV16 PsV in cell-based assays. Serum LNG was at levels associated with local contraceptive effects. The results demonstrate proof-of-concept of a novel core-matrix IVR for sustained and simultaneous delivery of diverse molecules for the prevention of HIV, HSV-2 and HPV acquisition, as well as unintended pregnancy.

A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.

In vitro and in vivo evaluation of two carrageenan-based formulations to prevent HPV acquisition.

Commercial vaccines against human papillomavirus (HPV) have low uptake due to parental autonomy, dosing regimen, cost, and cold chain storage requirements. Carrageenan (CG)-based formulations prevent HPV infection in vitro and in vivo but data are needed on the durability of anti-HPV activity and the effect of seminal plasma (SP). The Population Council's PC-515 gel and the lubricant Divine 9 were tested for their physicochemical properties and anti-HPV activity against HPV16, 18, and 45 pseudoviruses (PsVs). Anti-PsV activity was estimated using the luciferase assay in HeLa cells and the HPV PsV luciferase mouse model. Formulations were applied intravaginally either 2h pre/2h post (-2h/+2h) or 24h pre (-24h) relative to challenge with HPV16 or 45 PsV in PBS or SP/PBS. Both formulations showed broad-spectrum anti-HPV activity in vitro (IC50: 1-20ng/ml), significantly decreasing HPV PsV infection in the mouse model (-2h/+2h, p<0.0001). PC-515 protected better than Divine 9 in the -24h dosing regimen (p<0.0001) and comparable to Divine 9 in the -2h/+2h regimen (p=0.9841). PC-515 retained full activity in the murine model when PsV solutions contained human SP. The durable, potential broad-spectrum anti-HPV activity of CG formulations in the presence of SP supports their further development to prevent HPV acquisition.

A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV.

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 10(6) pfu HSV-2 were applied immediately after vaginal challenge and also when 5×10(3) pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×10(6) HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.

Exposure to MIV-150 from a high-dose intravaginal ring results in limited emergence of drug resistance mutations in SHIV-RT infected rhesus macaques.

When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.

MIV-150-containing intravaginal rings protect macaque vaginal explants against SHIV-RT infection.

Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs.

A modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus 2 infection in vivo.

We previously showed that a prototype gel comprising zinc acetate (ZA) in carrageenan (CG) protected mice against vaginal and rectal herpes simplex virus 2 (HSV-2) challenge as well as macaques against vaginal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge. In this work, we modified buffers and cosolvents to obtain a stable, nearly iso-osmolal formulation and evaluated its safety and efficacy against SHIV-RT and HSV-2. In vitro toxicity to lactobacilli and Candida albicans was determined. Macaques were given daily doses of ZA and CG (ZA/CG) or CG alone vaginally for 14 days and challenged with SHIV-RT 24 h later. Mice were challenged vaginally or rectally with HSV-2 immediately after a single gel treatment to measure efficacy or vaginally 12 h after daily gel treatment for 7 days to evaluate the gel's impact on susceptibility to HSV-2 infection. The modified ZA/CG neither affected the viability of lactobacilli or C. albicans nor enhanced vaginal HSV-2 infection after daily ZA/CG treatment. Vaginal SHIV-RT infection of macaques was reduced by 66% (P = 0.006) when macaques were challenged 24 h after the last dose of gel. We observed 60% to 80% uninfected mice after vaginal (P < 0.0001) and rectal (P = 0.008) high-dose HSV-2 challenge. The modified ZA/CG gel is safe and effective in animal models and represents a potential candidate to limit the transmission of HIV and HSV-2.

An intravaginal ring that releases the NNRTI MIV-150 reduces SHIV transmission in macaques.

Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150-containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs.

Zinc acetate/carrageenan gels exhibit potent activity in vivo against high-dose herpes simplex virus 2 vaginal and rectal challenge.

Topical microbicides that block the sexual transmission of HIV and herpes simplex virus 2 (HSV-2) are desperately needed to reduce the incidence of HIV infections worldwide. Previously we completed phase 3 testing of the carrageenan-based gel Carraguard. Although the trial did not show that Carraguard is effective in preventing HIV transmission during vaginal sex, it did show that Carraguard is safe when used weekly for up to 2 years. Moreover, Carraguard has in vitro activity against human papillomavirus (HPV) and HSV-2 and favorable physical and rheological properties, which makes it a useful vehicle to deliver antiviral agents such as zinc acetate. To that end, we previously reported that a prototype zinc acetate carrageenan gel protects macaques against vaginal challenge with combined simian-human immunodeficiency virus reverse transcriptase (SHIV-RT). Herein, we report the safety and efficacy of a series of zinc acetate and/or carrageenan gels. The gels protected mice (75 to 85% survival; P < 0.001) against high-dose (10(6)-PFU) HSV-2 vaginal or rectal challenge. In contrast, zinc acetate formulated in HEC (hydroxyethylcellulose; or the Universal Placebo) failed to protect mice against the high-dose vaginal HSV-2 challenge (similar to aqueous zinc acetate solution and the placebo controls). The gels were found to be effective spreading gels, exhibited limited toxicity in vitro, caused minimal damage to the architecture of the cervicovaginal and rectal mucosae in vivo, and induced no increased susceptibility to HSV-2 infection in a mouse model. Our results provide a strong rationale to further optimize and evaluate the zinc acetate/carrageenan gels for their ability to block the sexual transmission of HIV and HSV-2.

An antiretroviral/zinc combination gel provides 24 hours of complete protection against vaginal SHIV infection in macaques.

BACKGROUND: Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application. METHODS AND FINDINGS: Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8-24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h. CONCLUSIONS: A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans.